This project will evaluate a novel strategy for rapid molecular confirmation of response to tuberculosis (TB) treatment. TB is a chronic infection caused by Mycobacterium tuberculosis complex (MTBC). The course of treatment is long and treatment failure is common. Because it can take months or years for sub-optimal treatment to manifest clinically in the form of recurrent disease, it is important to actively monitor treatment response by assessment of patient sputum samples during and at the end of TB treatment. Current methods for doing so are problematic. Microbiological culture is very slow to yield results, whereas rapid PCR-based tests for MTBC DNA in sputum are poor at distinguishing viable pathogen cells from inactivated ones, or from free nucleic acid fragments. In order to combine the speed of PCR with the specificity of microbiological culture for viable pathogen cells, we developed a new method termed Molecular Viability Testing. MVT correlates microbial viability with the ability to rapidly synthesize ribosomal RNA precursor (pre-rRNA) in response to a brief nutritional stimulus. Pre-rRNA is detected by measurement of species-specific pre-rRNA sequences in complex samples. Pre-rRNA in inactivated cells, and free nucleic acids, do not increase upon nutritional stimulation and are excluded. MVT is also highly sensitive. This R21 project will test the hypothesis that MVT conducted during TB treatment is more accurate than standard PCR. Specific aims are 1) to characterize the analytical performance of the MVT assay using laboratory-generated samples and archived patient samples, and 2) to test the hypothesis that MVT is significantly more accurate than Cepheid GeneXpert Ultra qPCR, relative to a MTBC culture gold standard, when applied to samples collected from South African patients over the course of TB treatment. If successful, this project will improve our ability to confirm TB cure and facilitate clinical evaluation of new TB drugs and vaccines.